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Figure 5


Fig. 5. Centromeric localization of INCENP and aurora B is fragmented in cohesin-depleted spindles. (A) Fluorescence images showing DNA (blue), tubulin (red) and Ndc80, INCENP or aurora B (green) in spindles formed in cycled extracts in the presence ({Delta}Mock) or absence ({Delta}Cohesin) of cohesin. Reactions were spun onto coverslips 60 minutes into metaphase. Bar, 10 µm. (B) Fluorescence images of replicated, isolated sperm chromosomes assembled in mock- and cohesin-depleted extracts, spun onto coverslips 60 minutes after induction of mitosis and processed for immunofluorescence with antibodies to INCENP (green) and the kinetochore protein BubR1 (red). DNA was counterstained with Hoechst-33258 dye (blue). Bar, 2.5 µm. (C) Bipolar spindles assembled in the presence of Rhodamine-labeled tubulin (red) in control ({Delta}Mock) or cohesin ({Delta}Cohesin) depleted extracts were induced to enter anaphase, fixed after 20 minutes and spun onto coverslips for immunofluorescence using antibodies against INCENP and aurora B (green). DNA was counterstained with Hoechst-33258 dye (blue) and microtubules are shown in red. Bar, 10 µm.